Reactive Oxygen Species Regulate Oxygen-Sensitive Potassium Flux in Rainbow Trout Erythrocytes

In the present study, we have investigated if reactive oxygen species are involved in the oxygen-dependent regulation of potassium-chloride cotransport activity in trout erythrocyte membrane. An increase in the oxygen level caused an increase in chloride-sensitive potassium transport (K⁺-Cl⁻ cotransport). 5 mM hydrogen peroxide caused an increase in K⁺-Cl⁻ cotransport at 5% oxygen. The increase in flux could be inhibited by adding extracellular catalase in the incubation. Pretreatment of the cells with mercaptopropionyl glycine (MPG), a scavenger of reactive oxygen species showing preference for hydroxyl radicals, abolished the activation of the K⁺-Cl⁻ cotransporter by increased oxygen levels. The inhibition by MPG was reversible, and MPG could not inhibit the activation of transporter by the sulfhydryl reagent, N-ethylmaleimide, indicating that the effect of MPG was due to the scavenging of reactive oxygen species and not to the reaction of MPG with the cotransporter. Copper ions, which catalyze the production of hydroxyl radicals in the Fenton reaction, activated K⁺-Cl⁻ cotransport significantly at hypoxic conditions (1% O₂). These data suggest that hydroxyl radicals, formed from O₂ in close vicinity to the cell membrane, play an important role in the oxygen-dependent activation of the K⁺-Cl⁻ cotransporter.